Programmed Cell Death is the process by which cells die as part of the normal development and homeostasis of the organism. Apoptosis is the genetically controlled process by which most programmed cell deaths occur in living organisms. Malfunction of the carefully regulated apoptotic program has been found to contribute to the pathogenesis of human diseases such as AIDS, cancer, neurodegenerative and autoimmune diseases. Bcl-2 and Bcl-xL are two proteins known to be very powerful repressors of apoptosis. Many aspects of the regulation of the anti-apoptotic activities of these two proteins remain obscure. There is evidence that the regulation of proteins involved in apoptosis is achieved via interaction with other proteins. To identify proteins that might bind Bcl-2 or Bcl-xL, and therefore regulate their functions, two-hybrid screenings were carried out using Bcl-2 and Bcl-xL as baits. One novel protein, BBP-1, was identified that binds both Bcl-2 and Bcl-xL. The hypothesis of this research project is that binding of BBP-1 to Bcl-2 and Bcl-xL regulates the activities of these two proteins. To test this hypothesis, the function and biochemistry of BBP-1 must first be characterized. The information obtained will set the stage for future studies, in which the regulatory effect of BBP-1 on the anti-apoptotic activities of Bcl-2 and Bcl-xL will be analyzed in detail. During the present funding period an antibody has been produced against BBP-1, its pro-apoptotic activity has been detected in an additional cell line, Bcl-2 has been shown to protect from BBP-1 induced killing, and data has been obtained on BBP-1's tissue expression. The aim of this research project is to continue and extend the characterization of BBP-1 that is currently being performed. Experiments are proposed to study the binding of BBP-1 to Bcl-xL in mammalian cells, identify the domains that are responsible for BBP-1/Bcl-2 and BBPl/Bcl-xL interactions, and determine the native structure of BBP-1. BBP-1 localization studies will be expanded using immunohistochemistry analysis, and experiments will be performed to establish how the proapoptotic activity of BBP-1 is modulated. Further efforts will be directed to the identification and characterization of other proteins that bind to BBP-1 and additional proteins that interact with Bcl-xL. The results of these studies should enhance our understanding of the apoptosis process and its regulation, which should identify novel targets for the development of therapeutic agents that could be used in the future to treat and/or prevent human diseases characterized by abnormal levels of cell death.